This paper presents a new computer method for folding an RNA molecule that finds a conformation of minimum free energy using published values of stacking and destabilizing energies. It is based on a dynamic programming algorithm from applied mathematics, and is much more efficient, faster, and can fold larger molecules than procedures which have appeared up to now in the biological literature. Its power is demonstrated in the folding of a 459 nucleotide immunoglobulin gamma 1 heavy chain messenger RNA fragment. We go beyond the basic method to show how to incorporate additional information into the algorithm. This includes data on chemical reactivity and enzyme susceptibility. We illustrate this with the folding of two large fragments from the 16S ribosomal RNA of Escherichia coli.

Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.

Extensive studies in our laboratory using different ribonucleases resulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies have precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coli 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.

A consensus on the folding of the Escherichia coli 16-S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available. We therefore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E. coli 16-S RNA and other ribosomal RNAs. All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E. coli molecule. This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs. The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the ribosome.The universal function of ribosomes is to decode the genetic message and to catalyze peptidyl bond formation. Since the sequence of ribosomal RNAs appears to change very slowly in the course of evolution, Woese and his collaborators have undertaken phylogenetic comparison from extensive analysis of T I ribonuclease-generated oligonucleotides of the small ribosomal subunit RNA from a large variety of organisms and organelles [I -41. These studies provided the first evidence for the existence of nucleotide sequence homologies among the various molecules, reflecting the strong evolutionary pressure imposed by their basic function. It seems likely that the secondary structure of the ribosomal RNA molecule (or at least part of this structure) is highly constrained as well as already observed for tRNA and 5-S RNA molecules.At present, several complete nucleotide sequences of small ribosomal subunit RNAs have been determined. They cover diverse types of organisms or organelles: (a) bacterial 16-S RNAs from Escherichia coli [5, 61 and Proteus vulgaris [7], (b) chloroplastic 16-S RNA from Zea mays [S], (c) cytoplasmic 18-S RNA from Succhuromyces cerevisiae [9] and Xenopus laevis [lo], (d) mitochondrial 12-S RNAs from human placenta [Ill and mouse [12], and mitochondrial 15-S RNA from yeast (S. cerevisiae) [I 31. A consensus on the folding of the E. coli 16-S RNA, supported by phylogenetic and topographical data, is now emerging . There is strong evidence that bacterial 16-S RNAs are organized in a common folding pattern [7, 141. In this paper, we investigate the extent of similarities and dissimilarities, in terms of primary and secondary structures, between E. coli 16-S RNA and the other ribosomal RNAs from evolutionary distant species. We find a rather high degree of sequence conservation. Furthermore, a large number of secondary structure elements proposed for E. coli 16-S RNA appear to be conserved in those of the other species. Some of them are even preserved in all the molecules examined so far and are therefore good candidates for being universal structures. In addition, nucleotide residues are strictly conserved at eq...

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation, or oxidants. The resulting formation of protein-bound poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA.

The RNA binding sites of four 30-S ribosom24 subunit proteins from Escherichia coli, namely S4, S8, S15 and S20 were prepared from reconstituted single protein . 36-S-RNA complexes by mild enzymic digestion of non-protected RNA regions. Oligonucleotide fingerprints of the protected RNA regions were obtained and their positions were located within the 16-S RNA sequence. They were not completely contiguous regions of RNA ; oligonucleotides had been excised from each of them. The binding sites of S4 and S20, and those of S8 and S15 showed overlapping. The specificity of the RNA binding sites was confirmed by a reconstitution method.Four proteins have been identified that bind strongly to 16-S RNA, namely S4, S8, S15 and S20. Others, including S7 and S17, bind less strongly [l -51. Recently, attempts have been made at characterizing the RNA binding sites of these proteins. The methods have involved either the binding of proteins to RNA fragments obtained by mild digestion of 16-S RNA, or the controlled ribonuclease digestion of single protein . RNA complexes. The former method has been mainly used by Zimmermann et al. [ 5 ] , to give partial localisation of the protein binding sites on 16-S RNA and to show that the RNA binding proteins S4, S8, S15 and S20 attach to the 5'-half of the 16-S RNA. Using the latter method, Kurland and colleagues [6,7] have obtained a comparable result for S4 and a more precise localisation of the S8 binding site.In this study, an electrophoretic method was employed to isolate single protein . RNA fragment complexes. Thereby, protected RNA regions were obtained that corresponded to the binding sites of each of the proteins S4, S8, S15 and S20. The recent completion of most of the 16-S RNA nucleotide sequence [8 -101 permitted a precise location of these binding sites within the RNA molecule. Moreover, a theoretical evaluation of a base-pairing scheme for

Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.

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